Survey on the prevalence of intestinal parasites in domestic cats (Felis catus Linnaeus, 1758) in central Nepal

Abstract Introduction Cats (Felis catus) are the only felines that live in close contact with humans. Since cats can act as vectors, carriers, reservoirs and definitive hosts of many gastrointestinal (GI) parasites, parasitic assessment could contribute to their survival and well‐being. Aims The current study aimed to assess the diversity and prevalence of GI parasites in domestic and feral cats from Ratnanagar in Chitwan in Central Nepal. Methods A total of 107 fresh faecal samples of cats (90 household cats and 17 feral cats) of varied ages and sex were collected and transported to the laboratory. The copromicroscopic examination was carried out following direct wet mount, formalin‐ethyl acetate sedimentation, saturated salt flotation, acid‐fast staining and sporulation techniques. Furthermore, associated risk factors were evaluated to ascertain the predictor of risks for parasitic acquisition. Results The current study revealed an overall 95.3% prevalence rate with a 100% rate in feral cats and 94.4% in household cats. Altogether, 18 (17 known and one unknown) different species of GI parasites were reported with the helminths (95.3%; 11 species) and the protozoa (55.1%; seven species). Besides age and sex, outdoor lifestyle, absence or unknown history of medication and hunting behaviour of the felines are the predictors of risk. Furthermore, mixed infection was comparatively higher than single infection in the faecal samples. Conclusions Cats harbour a higher prevalence and greater diversity of GI parasites, and parasitism varies with age and sex. This finding can be essential for veterinarians and public health authorities for strategic treatment and for assessing the zoonotic transmission of the parasites from these felines. Importantly, an effective medication strategy for cats and owners is recommended.


INTRODUCTION
Domestic cats (Felis catus Linnaeus, 1758) with Taxonomic Serial Number 183798 (www.itis.gov) are the only felines living exclusively in close contact with humans. Ecologically, they are the world's most invasive species (Medina et al., 2011) and can be found in almost every terrestrial habitat (Trouwborst et al., 2020).
Because of their ability to hunt rodents and for companionship, these mammals are mostly domesticated as pets (Hill, 2008). However, they are reared as a mouser in agricultural regions in rural areas.
In addition, the presence of infected cats around human inhabitants may pose a severe public health concern. However, the prevalence and diversity of GI parasites in cats in Nepal have not been fully explored yet. Therefore, the objectives of the current study were to determine the diversity and prevalence of GI parasites among domestic and feral cats and to analyse how these parasites could affect feline health. These findings will be important for treating parasites in cats and controlling possible zoonosis.

Study area
The current study was conducted in Ratnanagar Municipality in eastern Chitwan in Central Nepal ( Figure 1

Sample collection, preservation and transportation
The faecal sampling had been conducted from 2 May to 30 November 2020. For household cats, we employed the door-to-door visit method. For convenience, the owners/farmers were instructed to keep their cats indoors overnight and the collection was performed the next morning. The details on age, sex, medication and rearing practices adopted were noted. Furthermore, opportunistic selection of the faecal samples was employed for feral cats. Since feral cats are pretty shy in nature, they avoid crowded environments and are likely to bury their faeces. Thus, they were followed quietly until they defecate. Furthermore, artificial sand trays/boxes were also placed in different core sites.
By nature, feral cats are highly suspicious to any things new, so we further kept some foods like bread in and around such trays to attract them to it and waited until they accept these trays and defecate over there. The stool samples were also collected from the gardens, edges of roads and agricultural fields. Finally, 107 fresh faecal samples (n = 107) were collected that included 90 faeces from household cats (n1 = 90) and 17 faeces from feral cats (n2 = 17). Their population was classified into two groups: young: ≤ 12 months of age and old/adult cats: > 12 months based on the direct observation of the fresh faecal pellets. The collected faecal samples were carefully observed for their consistency and the presence of blood, mucus, adult nematodes and detached segments of cestodes in the sampling sites and then simultaneously preserved in 2.5% weight/volume (w/v) potassium dichromate solution in 20 ml sterile vials. The samples were then transported to Animal Research Laboratory, Faculty of Science, Nepal Academy of F I G U R E 1 Map of study area Science and Technology, Lalitpur, Nepal, for additional investigation and microscopic observation.

Laboratory processing and examination
The copromicroscopic examination of the faecal samples was carried out with five different routine laboratory techniques like direct wet mount, formalin-ethyl acetate sedimentation, saturated sodium chloride salt flotation (45% w/v NaCl), acid-fast staining and sporulation assays as previously explained Adhikari et al., 2022;Aryal et al., 2021;Ghimire & Bhattarai, 2019;Ghimire et al., 2021).
For direct wet mount, simply a drop of well-mixed faecal sample in 2.5% potassium dichromate was kept on the glass slide and then observed under the microscope.
For formalin-ethyl acetate sedimentation, 2 gm of the faecal sample was mixed with normal saline (0.85% NaCl) in a 15 ml centrifuge tube. It was then centrifuged at 1200 rpm for 5 min and the supernatant was discarded. The sediment was further added with 10 ml 10% formalin and 4 ml ethyl acetate and re-centrifuged (1200 rpm X 5 min) for better separation of debris. Finally, the supernatant layers were discarded, and a single drop of the sediment was taken for microscopic observation.
For the flotation technique, the sediment obtained after the first centrifugation was added with 12 ml of 45% w/v NaCl (floating media) and re-centrifuged (1200 rpm X 5 min). Further, the floating media was added drop by drop until the tube was completely filled and a cover slip was placed at its mouth, such that the coverslip touched the floating media. After 10 min, the coverslip was carefully removed for microscopic observation.
Finally, for acid-fast staining, the sediment after formalin-ethyl acetate sedimentation was conducted for thin smear preparation. The smears were dried at room temperature and other procedure follows the chronological order (a) fixation in absolute methanol for 2 min, (b) counter-staining with carbol fuchsin for 15 min that follows destaining with acid alcohol and (c) restaining with malachite green for a minute that followed a gentle wash with distilled water. Finally, the smears were dried and observed under the microscope with immersion oil.

Parasite identification
gondii and Hammondi hammondi) are very difficult to detect morphologically as well as serologically (Yang & Liang, 2015), we named them T. gondii/H. hammondi.

Data analysis
All the data generated were expressed as numbers of positive samples and prevalence rates in the

RESULTS
The macroscopic observation was carried out in all the faecal samples (n = 107) that had been collected from the sampling sites. Five different types of faecal samples were recorded. The study of faecal consistency found 46 formed stool (43%), 33 mixed stool (30.8%), 14 half-formed and half diarrhoeal (13.1%), 19 mainly formed and few softer stool (17.8%), 16 mushy and lumpy stool (15%), seven hard-constipated stool (6.5%) and five diarrhoeal stool (4.7%). Except for five stool samples of formed consistency, all rest stool types were positive for parasites (Supporting Informations 2 and 3).
Importantly, 16 different species (protozoa: six and helminths: 10) were reported in household cats and 16 different species (protozoa: seven and helminths: nine) were reported in feral cats aggregating a total of 18 different species (protozoa: seven and helminths: 11) among the overall population (Supporting Information 4; Table 1). The overall prevalence of helminths (92.5%) was significantly higher than that of protozoa (55.1%). Interestingly, three morphotypes of strongyle eggs The prevalence of each 17 parasitic species was compared with that reported in different countries (Table 2).
Considering the age, adult cats had a higher prevalence rate (98.6%) of enteric parasites than young cats (88.6%); however, the data were not significant (p > 0.05). The adults also harboured a more diverse parasite species than the youngs (18 vs. 12 species). Interestingly, Cryptosporidium and Toxocara were dominant in the young cats, whereas ancylostomatids and taeniids were dominant in the adults. Uniquely, Capillaria, Dipylidium, Toxoascaris, Hymenolepis, Archiacanthocephala and an unknown coccidian were found in the adults (Table 1; Figure 2).
Interestingly, regarding the rate of total parasites as well as protozoa, male cats had a higher rate than females; however, the data were not significant (p > 0.05). Further, males had a significantly higher prevalence of helminth species, compared to females (p < 0.05). The rate was also higher in young males, compared with young females (95% vs. 80%) or in adult males, compared with adult females (100% vs. 97.9%; Table 1). Besides age and sex differences, other possible predictors of risks like lifestyle, medication and feeding behaviour of the cats were also analysed. Parasitic prevalence was significantly higher in those populations that exclusively live outdoors, have unknown or no history of medication and those that feed exclusively by hunting (Table 3).
Concurrency analysis showed that most cats possessed multiple parasite infections, compared to single infections (87.9% vs. 7.5%).
The young cats contained up to four species, whereas the adult cats contained up to six species of parasites (Table 1).
The presence of parasites with respect to specific faecal consistency was also analysed. Cystoisospora spp., Cryptosporidium sp., and A. tubaeforme were dominant in diarrhoeal stools. In contrast, T. cati and A.
tubaeforme were usual in all types of stools (Supporting Information 5).

DISCUSSION
As per our knowledge, this is the first peer-reviewed international report regarding the comparative diversity and prevalence of GI par-  Ngui et al., 2014;Tun et al., 2015). Such discrepancy in prevalence rates might be attributed to various factors like rural inhabitation, outdoor access, tropical to subtropical climatic conditions, poor feeding practices and unregulated deworming (Chalkowski et al., 2019;Genchi et al., 2021;Mircean et al., 2010).
In the current study, the prevalence of helminth parasites was higher than that of protozoa. This finding is per the outcomes from Italy (Mircean et al., 2010) and Malaysia (Ngui et al., 2014). Ancylostoma spp.
In addition, most of the current felines have easy outdoor access.
Thus, these two factors might have cumulatively increased the greater acquisition success of ancylostomatidae worm resulting in their higher prevalence. Similar to it, the current geo-climatic condition also favours the quick development of unsegmented ova of ascarid into infective form in the soil. Interestingly, the prevalence of T. cati was higher than T. leonina. This discrepancy might have occurred due to the variance in their transmission route. Apart from ingestion of embryonated eggs and tissue of paratenic hosts (e.g., rodents, earthworms, cockroach and birds) by cats, newly born kitten acquire T. cati via pre-and perinatal route, but T. leonina does not follow this route and rarely occurs in kittens (Parsons, 1987). Epidemiologically, the shedding of a large number of eggs in each defecation contributes to the dissemination of the infection to other similar hosts and humans in the surrounding periphery, indicating that a large number of infected cats nearby human habitation can be a matter of public health concern. Furthermore, cats are also known to harbour several strongylid species like Aelurostrongylus abstrusus, A. chabaudi, Angiostrongylus vasorum, Troglostrongylus brevior, T. subcrenatus (Brianti et al., 2012;Giannelli et al., 2017;Gueldner et al., 2019;Jefferies et al., 2010;Mircean et al., 2010;Varcasia et al., 2014), and Strongyloides spp. like Strongyloides felis, S. planiceps, S. stercoralis and S. tumefaciens (Wulcan et al., 2019). However, we could only document three morphotypes of strongylid eggs and two morphotypes of Strongyloides eggs. Therefore, a detailed molecular study of these nematodes should be conducted in the future. canis, Pulex irritans) and lice (Trichodectes canis) by the current felines (Rousseau et al., 2022). In the same way, the hunting behaviour and preference of rodents, terrestrial insects and myriapods might have contributed to the acquisition of Hymenolepis sp. and Archiacanthocephala (Morand et al., 2007).
Regarding the protozoa, Cystoisospora spp. (Cystoisospora felis and C. rivolta) were the most dominant GI parasites. Since kittens are immunologically weak, they are highly susceptible to cocciodiosis (Dubey, 2018). An infected kitten can be contagious to other members of its group, and their contact with the mother's faeces may be a risk. Surprisingly, the presence of other coccidia like Cryptosporidium, T. gondii/H. hammondi and Sarcocystis spp. might be critical because these coccidia possess zoonotic potentiality, especially in the immunocompromised host (Shukla et al., 2006;Torda, 2001). Regarding it, a case of congenital toxoplasmosis had also been reported in Nepal in a 53-day-old baby who died within 6 days after the diagnosis and beginning of treatment (Rai et al., 2011) (Bowman, 2014;Dubey, 1976;Soulsby, 2012), we could record four different morphotypes. This indicates an urgent need for further study that clarifies the taxonomic position of these coccidia in Nepalese cats.
Regarding Sarcodina, the presence of Entamoeba sp. (15%) has been distinctly noted although amoebiasis is usually uncommon in cats (Josephine, 1958). Epidemiologically, amoeba could be higher in those hosts that are rural inhabitants with poor lifestyles and feeding practices (Nath et al., 2015), which might be justifiable for their presence in the current felines.
Regarding sex-wise bias, our report of higher prevalence in male cats contrasts, with those from Europe (Giannelli et al., 2017), Italy (Mircean et al., 2010) and Korea (Lee et al., 2019). The female cats usually remain within their home ranges, while the adult males are the free roamers.
On attaining sexual maturity, male cats typically leave their home and become feral (Bikana Chaudhary personal communication, November 8, 2020). Besides, felines have promiscuous mating systems, that is, a single male mates with several females (Natoli et al., 2000). Thus, owing to the intimacy or proximity and higher contact rates with multiple sex partners, the possibility of the acquisition/transmission of parasites by male felines cannot be ignored (Altizer et al., 2003). Importantly, the testosterone hormone acts as an immune suppressor (Salvador et al., 1996), resulting in the susceptibility to the parasitic infection, although this should be checked further.
Adult cats usually roam in and around their home ranges (door-todoor, agricultural fields, roads and animal sheds) for food or mates, death of a young Romanian cat (Giannelli et al., 2017). However, a feral cat infected with multiple parasite species had lesser Toxocara spp. load (Serrano & Millán, 2014) suggesting the probable negative or positive effects of polyparasitism in the hosts (Hoarau et al., 2020). The detection of the majority of Cystoisospora spp. and Cryptosporidium sp.
in diarrhoeal stool and A. tubaeforme in diarrhoeal, mushy and lumpy, mixed, formed, and hard-constipated stool indicates that these GI parasitism results in different pathologies in the hosts. Because the current study lacks histopathologic assays, further studies should confirm the causal association of coinfection in the host survival.
The current study is subjected to mainly two limitations, which should be considered. First, assessment of only a single faecal sample from a single feline and employment of smear assessment alone for parasitic determination might not be enough to accommodate the pre-patent infections and the intermittent shedding of the parasitic cyst/eggs/oocysts/larva. It may underrate the quantification of prevalence as well as the concomitance of the parasites. A molecular study would support and elaborate the species-level identification of the parasites. Second, the cross-sectional method and use of a small and unbalanced sample size (n = 107) of domestic and feral cats may create difficulty in discussing the reason behind the discrepancy. However, we have strictly secured the quality control during field surveys and laboratory techniques, which we believe is the first copromicroscopic study in Nepal to address the unique GI parasites in domestic and feral cats.

CONCLUSION AND RECOMMENDATIONS
In conclusion, cats in the study area harbour a high prevalence of diverse GI parasites that vary according to age and sex. They are also associated with the predictors like outdoor lifestyle, absence of medication and hunting behaviour. The presence of major zoonotic parasites like Ancylostoma, Cryptosporidium, Sarcocystis, Toxocara, Toxoplasma and Strongyloides in the current cat populations indicates that the local peo-ple might be at risk of public health concerns. Therefore, an awareness programme for the local people and cat owners should be conducted for bringing up the treatment interventions, healthy feeding practices and proper disposal of cat faeces. Further, a One Health approach accompanied by molecular studies of faecal samples of cats, humans and domestic animals should be conducted to clarify the existence of the zoonotic epidemiology of the GI parasites.

DATA AVAILABILITY STATEMENT
The data that supports the findings of this study are available in the supplementary material of this article.

ETHICS STATEMENT
The required permission for collecting the faecal samples was issued by Ratnanagar Municipality and Ratnanagar Municipality Veterinary Service (Chitwan, Nepal;Permission No. 498/2077-78). The study had been conducted using the stool samples defecated at the surface, and no experimental infection in cats was established during the work.